Selection of explants (1) Generally speaking, explants in tissue culture can be divided into two types: one is explants with buds, such as shoot tips, lateral buds, scale buds and protocorms. It can directly induce and promote the production of clustered buds in the process of tissue culture, and the success rate of obtaining regenerated plants is high, the variability is small, and it is easy to maintain the excellent properties of materials. The other is mainly the vegetative organs such as roots and leaves and the reproductive organs such as anthers, petals, receptacle, ovules and fruits. Most of these explants need a dedifferentiation process, which differentiates into buds or embryoids after callus stage, and then forms regenerated plants.
In rapid propagation, the most commonly used explant is the stem tip. Usually, the incision is about 0.5 cm, which is too small to produce callus. If it is too big, it will take up too much space in the culture container. If virus-free seedlings are cultured by explants, only the meristem of the shoot tip is usually taken, and its length is usually below 0.1mm.
(2) Sterilization of explants Because most explants use plants that grow from the outside, they often carry various microorganisms. If they are brought into the culture medium, they will multiply rapidly, form pollution and lead to culture failure. Therefore, the disinfection of explants is an essential work. Disinfection should not only kill the germs on the explants, but also damage the materials and affect their growth. Because of different materials, different culture conditions and seasons, different explants should be disinfected with their own suitable disinfectant types, disinfectant concentrations, disinfection time and treatment procedures.
The ideal disinfectant should have strong bactericidal ability, and should be easy to remove and not easy to damage explants. Commonly used are sodium hypochlorite (0.5% ~ 10%) and bleaching powder (1% ~ 10%) filtrate, which can decompose to produce chlorine gas with bactericidal effect and emit it into the air by itself. As long as the concentration is appropriate, it will generally not hurt the explants. Hydrogen peroxide (3% ~ 10%) is also easy to decompose and not easy to damage explants. Alcohol (70%) has strong permeability, bactericidal effect and is volatile, but it will kill tissues and cells, so the disinfection time should not be too long. Commonly used alcohol is disinfected for a few seconds first, which is beneficial for other disinfectants to penetrate into the material to play a bactericidal role.
Disinfection methods and procedures vary with different materials. Usually soak it in alcohol (70%) for several seconds, then take it out and soak it in 10% calcium hypochlorite saturated supernatant 10 ~ 20 minutes or 2% ~ 10% sodium hypochlorite solution for 6 ~ 15 minutes, and then rinse it with sterile water for three times.
2. Proliferation of explants
The proliferation of explants is the key stage of tissue culture. After inoculation, the culture container is in the culture room. Generally, light 16 hours per day, 1500 ~ 3000 lux, and the temperature is about 25℃ for differentiation culture. In order to expand the reproductive coefficient after the formation of new buds, subculture is needed. The material is divided into plants or slices and transferred to the proliferation medium, which is generally improved on the differentiation medium to improve the proliferation rate. After 1 month multiplication culture, it can be proliferated again according to the situation, and the number of plants can be increased after subculture.
In subculture, because the explants themselves come from sterile environment, disinfection is not needed and the operation is convenient. But the differentiation ability of explants in subculture will gradually decrease, so subculture algebra is not endless.
3. Root induction
Adventitious buds and lateral buds formed by subculture generally have no roots and must be transferred to rooting medium for rooting culture. Rooting culture is mostly 1/2MS medium, because reducing the concentration of inorganic salts is beneficial to root differentiation. In addition, rooting medium and proliferation medium are very different in the types and concentrations of hormones, such as cytokinin and auxin. Generally speaking, cytokinin inhibits rooting, while auxin promotes rooting.
Generally, strong roots can be obtained by culturing in rooting medium for about 1 month. In addition, in production, there are also test-tube seedlings with root primordium, that is, they are only cultured in rooting medium for 7 ~ 10 days, and the root primordium or young roots less than 1 mm are induced before being transplanted. Because the basal incision has healed to form root primordium, it is not easy to be infected, and it takes root quickly after colonization and has a high survival rate.
4. hardening and transplanting of tissue culture seedlings
The test-tube seedlings that take root or form root primordium enter the natural environment from sterile and stable light, temperature and humidity environment, and the transformation process from heterotrophic to autotrophic must go through a domestication and exercise process, which is hardening seedlings.
Generally, before transplanting, the lid of the culture container is opened, and it is placed in natural light indoors for 3 days. Then, the seedlings are taken out, the agar in the roots is washed with tap water, and then planted in the prepared substrate. The matrix is usually peat, perlite, vermiculite, rice bran ash, etc. Or add some garden soil appropriately, and it is best to disinfect it with high temperature or drugs before use. In the early stage of transplanting, it is necessary to shade properly, strengthen water management, and maintain a high air humidity (about 90% relative humidity). But pay attention to the substrate should not be too wet, do not accumulate water to prevent rotten seedlings. In addition, temperature also has a great influence on the survival rate, and the optimum temperature is 15 ~ 25℃. In summer, when the temperature is too high and the water is low, the seedlings are easy to wither, and when the water is too high, they are easy to rot, which makes management difficult and the survival rate decreases. After 4 ~ 6 weeks of hardening, the new shoots can be transferred to normal management after they begin to grow.